Microgels for use in medical diagnosis and methods of making and using same

ABSTRACT

An electrophoresis microgel is formed in a gel holder. The gel holder comprises a top substrate, a bottom substrate and a spacer disposed between the top substrate and the bottom substrate. The spacer establishes a separation of from 25 to 250 microns between the top substrate and the bottom substrate. A gel compartment is formed by partially sealing the top substrate to the bottom substrate, while leaving an opening for the introduction of unpolymerized gel. The gel compartment is then filled with an unpolymerized gel, which is polymerized in the gel compartment. Electrodes may be printed on the substrates, may be contacts to an exposed edge of gel, or may be applied through windows cut into one of the substrates. One type of gel holder makes use of graded beads having a diameter of 25 to 250 microns slurried in an adhesive such as an acrylate adhesive as the spacer. The slurry is printed onto the surface of one or both substrates to form a spacer of the desired shape, and then hardened using heat or light. If desired, the spacer can establish lanes within the gel.

This application is a continuation-in-part and divisional of U.S. patentapplication Ser. No. 332,577 filed May 1, 1994, which is now U.S. Pat.No. 5,627,022.

BACKGROUND OF THE INVENTION

This application relates to microgels for use in medical diagnosis,especially for the sequencing of nucleic acids, and to methods of makingand using such gels.

DNA sequencing may be carried out using automated systems designed forlaboratory application. Methods and apparatus for sequencing of DNA aredescribed in U.S. Pat. Nos. 4,811,218; 4,823,007; 5,062,942; 5,091,652;5,119,316 and 5,122,345, which are incorporated herein by reference.

The general methodology employed in these systems involves breaking upthe sample DNA using restriction endonucleases; amplifying (for examplewith PCR) the restriction fragment of interest; combining the amplifiedDNA with a sequencing primer which may be the same as or different fromthe amplification primers; extending the sequencing primer in thepresence of normal nucleotide (A, C, G, and T) and a chain-terminatingnucleotide, such as a dideoxynucleotide, which prevents furtherextension of the primer once incorporated; and analyzing the product forthe length of the extended fragments obtained. Analysis of fragments maybe done by electrophoresis, for example on a polyacrylamide gel.

International Patent Publication No. WO93/00986 describeselectrophoresis gels with a thickness of 25 to 250 microns. The gels areformed between two clamped-together plates, one of which is grooved to adepth equal to the desired gel thickness to form parallel tracks whichare then filled with gel.

In performing a nucleic acid sequence analysis on a gel, thecharacteristics of the gel, including the size and thickness, impact thetime and cost required to do the analysis. Since it is desirable toreduce the time and cost of sequencing analyses in order to improve theavailable of sequencing as a diagnostic tool, it would be advantageousto have a gel which permitted analysis of very small quantities ofoligonucleotide fragments in a short period of time. It is an object ofthe present invention to provide such a gel.

It is a further object of the invention to provide single-use,disposable gel holders which are readily filled with gel to provide agel for rapid analysis of small samples.

It is a further object of the present invention to provide a method ofmaking gels which achieve high resolution of oligonucleotide fragmentsin a short period of time.

It is a further object of the invention to provide a method ofevaluating a sample containing oligonucleotide fragments of variouslengths.

SUMMARY OF THE INVENTION

These and other objects of the invention are achieved using anelectrophoresis microgel formed in a gel holder. The gel holdercomprises a top substrate, a bottom substrate and a spacer disposedbetween the top substrate and the bottom substrate. The spacerestablishes a separation of from 250 micron or less, preferably 25 to250 microns, between the top substrate and the bottom substrate. A gelcompartment is formed by partially sealing the top substrate to thebottom substrate, while leaving an opening for the introduction ofunpolymerized gel. The gel compartment is then filled with anunpolymerized or partially polymerized gel, which is polymerized in thegel compartment. Electrodes may be printed on the substrates, may becontacts to an exposed edge of gel, or may be applied through windowscut into one of the substrates.

A preferred embodiment of the invention makes use of graded beads havinga diameter of 250 microns or less, preferably 25 to 250 microns,slurried in an adhesive such as an acrylic or ultraviolet-lightactivated adhesive or bonded to the substrate using a low-melting glassas the spacer. The slurry is printed onto the surface of one or bothsubstrates to form a spacer of the desired shape, and then hardenedusing heat or light. If desired, adhesive or a low-melting glasscontaining solid particles can also be used to establish lanes withinthe gel.

The spacing between the substrates can also be established using aplurality of beads disposed on the surface of the substrates within thegel compartment. In this case, the edges can be scaled by adhesive, orby adhesive-coated plastic films of appropriate thickness. Particles mayalso be disposed both within the adhesive and within the gelcompartment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exploded view of a first embodiment of a microgel inaccordance with the invention;

FIG. 2 shows a second embodiment of a microgel in accordance with theinvention;

FIG. 3 shows a third embodiment of the invention;

FIGS. 4A and 4B show a further embodiment of the invention;

FIGS. 5A and 5B show a gel forming insert useful in practicing theclaimed invention;

FIG. 6 shows a loading insert in accordance with the invention;

FIGS. 7A and 7B show further embodiments of loading and gel forminginserts in accordance with the invention;

FIG. 8 shows an apparatus for use in analyzing oligonucleotide mixturesin accordance with the invention;

FIG. 9 shows an apparatus for use in analyzing oligonucleotide mixturesin accordance with the invention;

FIG. 10 shows the results of an analysis of a mixture of a 30-mer and a31-mer using the present invention; and

FIGS. 11A-11F show the results of an analysis of the ddT sequencingreactions of M13 DNA template.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows an exploded view of a first embodiment of a microgel inaccordance with the invention. As shown, the microgel is formed from abottom substrate 1, a top substrate 2, and a spacer 3 sandwiched betweenthe two substrates. The spacer 3 and the interior surfaces of the twosubstrates 1,2 define a gel compartment 4. Top substrate 2 has a slot 5cut therein to permit loading of a sample onto the gel and slots 6 and 7through which electrodes can be placed in contact with the gel.

The microgel shown in FIG. 1 can be made by a "squash-filling" processas follows. First, the spacer 3 is adhered to the bottom substrate 1.The area of the gel compartment 4 is then filled with unpolymerized orpartially polymerized gel, and the top substrate 2 is pressed down overthe top. The unpolymerized gel fills the gel compartment 4, with anyexcess gel being squeezed out between around the edges of the substrate.The gel is then polymerized, and the excess is trimmed off.

FIG. 2 shows an exploded view of a second embodiment of a microgel inaccordance with the invention which is adapted to be filled by adifferent technique. In FIG. 2, the spacer 23 only extends along threesides of the bottom substrate 1, leaving an opening 24 along the fourthedge. As an alternative to the electrode slots 6 and 7 shown in FIG. 1,electrodes 26 and 27, may be deposited as a film directly on the top orbottom substrates. Suitable electrode materials are thin films ofconductive materials such as indium tin oxide (ITO) or platinum.Solution buffered electrodes, i.e. electrodes in which the electricalcontact between a conventional metallic (e.g. platinum) electrode andthe gel is indirect, and is mediated by a concentrated electrolytesolution, may also be used.

A microgel of the type shown in FIG. 2 can be made by first adhering thebottom substrate 1, the spacer 23 and the top substrate 2 together toform a gel holder assembly. The gel holder assembly is then placed in achamber and positioned vertically with the opening 24 directed downwardsover a reservoir of unpolymerized gel. The chamber is then evacuated,the gel holder assembly is lowered to immerse the opening in theunpolymerized gel, and a gas is reintroduced into the chamber atpositive pressure to drive the gel into the gel compartment 4 of the gelholder assembly. No slots are necessary in the top substrate of thisembodiment, because the gel is exposed through the opening 24,permitting edge loading of the gel, although one or more slots in thetop substrate may be provided if desired for introduction of sample orelectrodes.

A third embodiment of the invention is shown in FIG. 3. In this case,spacers have been used to create lane markers 30 within the body of thegel. Such lane markers may be incorporated to separate every lane, everyfourth lane, or at such other interals as may be preferred by the user.In a preferred configuration employing lane separators the gel isseparated into a number of lanes of equal width. The number of theselanes is determined by the formula (A/4), where A is the number ofuseable sample loading sites in the gel.

When the top substrate is shorter than the bottom substrate as shown inFIG. 3 it has been found to be advantageous to extend the lanes beyondthe end of the top substrate across at least a part of the extendingportion of the bottom substrate. This results in all lanes fillingindividually from the bottom up, and eliminates the possibility ofunpolymerized gel from one lane spilling over into an adjacent lane andcausing a bubble to form in the middle of that lane.

FIGS. 4A and 4B show a variation of a top substrate 2 with a window 5'cut therein to permit loading of sample. The edges of the window are allcut at 90°, except for the lower edge 31 which is cut with a 30° to 60°,preferably a 45° bevel, as shown most clearly in FIG. 4B. This designpermits improved sample loading.

In particular, a microgel using a top substrate cut as shown in FIGS. 4Aand 4B is preferably formed using a gel forming insert of the type shownin FIG. 5A. This insert has a base portion 1 and an insert portion 52.The insert portion 52 is notched at each end, such that it is narrowerthan the base portion. Preferably, the size of each notch is from 2-4 mmto 5 mm. The free edge of the insert portion is beveled to match thebevel formed in the lower edge 41 of the window 5', and the overall sizeof the insert is selected to permit insertion of the insert into thewindow 5'. For example, suitable dimensions are 12.5 cm wide, 1.5 cm intotal height and 1 mm in thickness. A tab or handle may be placed on onesurface of the base portion 41 to facilitate removal.

The gel forming insert is placed into the window as shown in FIG. 5Bprior to the polymerization of the gel, preferably prior to the fillingof the gel compartment. Adhesive tape may be used to constrain themovement of the gel forming insert so as to secure it against the bottomsubstrate and to prevent its floating on the injected solution. Uponinjection of the proper amount of polymerizable solution into themicrogel holder, the solution fills up the microgel compartment, up tothe gel forming insert, where the solution flows partly around the edgesof the gel forming insert, and it may bleed out of the microgel holderthrough the air holes. If there is a substantial excess of solutioninjected into the gel holder, the solution may even run out through thetop. The excess gel is not of any significant consequence to theeffective polymerization, sample loading, or use of the gel, and may betrimmed away if desired.

After UV catalyzed polymerization, it is important to the successful useof a microgel of the invention to delicately remove the gel forminginsert without damaging the gel. After polymerization, if the gel holderis tilted into its facial plane, the gel forming insert will tend tofall forward. Gentle tapping on the bottom plate may be necessary toencourage the displacement of the gel forming insert. Once an edge ofthe gel forming insert has emerged from the plane of the top substrate,it may be grasped and gently pulled away from any polymerized gel whichmay be in contact with the gel forming insert. Alternatively, adhesivetape attached to the outside face of the gel forming insert may be usedto pull the gel forming insert from its position. Either way, careshould be taken to preserve the even edge of gel which will have formedalong the bottom of the window of the top substrate.

Removal of the gel forming insert results in the formation of a troughrunning across the width of the gel bounded by the beveled edge of thewindow, the gel itself, and the bottom substrate into which the samplecan be inserted. It is into this trough which sample is loaded forelectrophoresis.

Because this trough is continuous across the width of the gel, it lacksthe slots normally used in the loading of sample onto a gel. It istherefore desirable to use a specially adapted loading insert of thetype shown in FIG. 6 when loading sample onto a gel formed as describedabove. The insert has a substantially rectangular base portion 61 havingtwo long edges and two short edges; and a plurality of fingers 62extending from one long edge of the base portion. Each of the fingerspreferably has a width of from 0.5 to 3 mm. The fingers are evenlyspaced at intervals of 2 to 7 mm in a region along the long edge of thebase portion staring and ending at a point from 2 to 4 mm from theadjacent short edge of the base portion so that the fingers fit withinthe trough. The fingers further have a 30°-60° bevel at the distal endthereof to match the bevel of the loading insert and the lower edge ofthe window. This insert is placed into the trough formed by the gelforming insert, and sample is then loaded into the gaps between thefingers.

A second and alternative method of preparing and loading a microgel,requires forming the microgel in the presence of a polymerization combinsert which has a series of flattened extensions which will form wellsfor the loading of sample on the gel. The polymerization comb insertreplaces the gel forming insert during the microgel filling andpolymerization process. The dimensions of the polymerization comb insertare identical to the gel forming insert, except that instead of having aflat bottom edge beveled at a 45 degree angle to be complementary to thebottom edge of the window in the top substrate, a notch of about 2 mmwidth and about 5 mm depth is made at intervals, e.g, every 7 mm alongthe bottom edge. The result is a row of teeth, each tooth having abottom edge beveled at 45 degrees to be complementary to the bottom edgeof the window in the top substrate. Once placed in the open window ofthe microgel holder, the polymerization comb insert may be sealed inplace with adhesive tape such that the entire window is completelycovered. An opening for the air holes is then introduced into theadhesive tape. Solution is then injected into the microgel holder. Thesolution fills the microgel compartment and flows into the gaps in theteeth of the Polymerization Comb. Excess solution bleeds out of the airholes. The solution may then be polymerized with UV induced catalysis asdescribed in the invention. When polymerization is complete, theadhesive tape and the polymerization comb insert are gently removed,leaving a row of gel columns vertically disposed against the bottomsubstrate. The gel columns act to define wells which can be used toseparate samples loaded by the conventional methods. Microgels are foundto have improved loading and using qualities if prior to theconstruction of the microgel holder, the gel compartment faces of thetop substrate and bottom substrate are treated with Bind Silane(Pharmacia).

FIGS. 7A and 7B shows alternative designs for filling plug and loadingcombs useful when the lane dividers are present on the extended portionof the bottom substrate (FIG. 3). In this case, grooves 101 are formedalong one surface of the inserts to accommodate the lane dividers.Advantageously, the grooves are slightly wider than the lane dividers toallow for air and excess gel to escape during the filling process.

The top and bottom substrates of the microgels of the invention arcpreferably transparent sheets of glass. For example 1 mm thicklow-fluorescing glass is a preferred material for use as the top andbottom substrate of the invention. 1 mm Borosilicate glass which hasgreater ultraviolet light transparency is another preferred material.Other materials such as plastics may also be used, however, providedthey do not fluoresce strongly in a region which would interfere withthe detection of a luminescent label on the nucleic acid polymer beingsequenced or inhibit the polymerization of the gel.

The spacers 3 in the microgels of the invention serve to create a gapbetween the top and bottom substrates to establish the gel compartment.In accordance with the invention, spacers are preferably selected suchthat this gap is between about 25 and 250 μm more preferably 25 to 100μm. Smaller gaps arc not really practical because of the greater caremust be taken during fabrication, for example the use of clean roomprocedures to prevent dust particles which may be large relative to thedesired gap from contaminating the surface of the substrate. Also, theextent to which both substrates are flat becomes more important as gapsize decrease. Lastly, the amount of sample loaded onto the gel may needto be reduced to avoid loss of the gels resolving ability which meansthat there will be a smaller emitted signal from the sample to detect.Gaps larger than 250 mm can be used, but may be less desirable becausethe electrical resistance of a gel of any given composition increases asthe gap size increases. This means that larger currents and hencegreater resistive heating of the gel will occur for a given electricfield strength in a thicker gel. Also, where the spacer is formed byapplying adhesive to one or both substrates, it is necessary to build upan adhesive layer that is essentially the same thickness as the gap. Itmay be difficult to find an adhesive which is viscous enough to bridgethe openings between spacers particles, has minimal flow out, permitssettling of the substrate onto the spacers and minimizes voids formed inthe application process.

One form of spacer useful in the invention is a polymeric film that isaffixed to the top or bottom substrate with an adhesive. For example,vinyl films coated with an acrylate adhesive can be cut to the desiredsize and pattern to form spacers. Suitable vinyl films include 1, 2 and4 mil films sold by 3M under the tradename SCOTCHCAL.

Spacers can also be formed by screen printing a slurry of particles in ahardenable material in the desired pattern onto the top or bottomsubstrate. For example, a UV-curable and/or thermally-curable acrylateadhesive slurried with graded silica particles of the appropriate sizeto define the desired gap between the substrates is a preferred materialfor use in forming a spacer by this mechanism. Suitable adhesivesinclude Loctite CHIPBONDER™# 346, 347, 360 and 3603, IMPRUV™ 349, 363,365 and 366 and General Purpose Ultraviolet Adhesive 352; and Minico®M07950-R acrylate adhesive.

An alternative to the use of an adhesive is to form a mixture frompowdered low melting glass and solid particles of an appropriate size toform the spacer. This mixture is screen printed onto the substrate inthe desired pattern, for example to form lanes within the gelcompartment, and then heated to melt the powdered low melting glass.Upon cooling, the low melting glass resolidifies, bonding the solidparticles to the substrate. In addition, the low-melting glass may beused alone or in conjunction with an adhesive to bond the top and thebottom substrates together.

The glass powder or "frit" will advantageously have a meltingtemperature of about 400° to 500° C., while the melting temperatures ofthe solid particles beads and the substrates will be form 600° to 800°C. Notwithstanding this difference in melting temperature, care shouldbe taken to match the thermal expansion coefficients of the glasspowder, substrates and solid particles to avoid fractures or voids dueto shrinkage on cooling. Acceptable results are achieved when thethermal expansion coefficients of the materials are within about 10% ofeach other.

Dispersions of particles in an adhesive can also be applied using arobotic glue gun which paints the adhesive onto the glass substrate inthe desired pattern. This has the advantage over screen printing thatnothing actually touches the substrate other than the adhesive.

Spacers can be formed using this approach on a single substrate. It ispreferred, however, to print matching patterns on both the top andbottom substrate that are aligned and pressed together to form thespacer between the two substrates. While this method introduces the needfor careful alignment, it has been found that it has the advantage ofreducing the formation of bubbles and voids in the spacer lines, thusleading to a more consistent product.

The spacing between the two substrates may be further defined andmaintained by a dispersion of solid beads within the gels itself. Thiscan be achieved by evenly spreading a slurry of beads, for example glassbeads of the appropriate size in a volatile solvent onto the interiorsurface of one of the substrates prior to the formation of the gelholder. The solvent is evaporated off, leaving the solid particlesdistributed on the surface of the substrate.

The gels of the invention offer the further advantage of highconsistency from one gel to the next. This advantages is principallyderived from the use of carefully controlled spacer thicknesses in amass production approach.

Microgels in accordance of the invention can be used to analyze quicklyand accurately a mixture of fluorescently-labeled oligonucleotidefragments of differing lengths. Basically, this process involves firstloading the mixture onto an origination site on a microgel in accordancewith the invention, and applying a potential to the electrophoresis gelwhereby a current flows through the electrophoresis gel and transportsthe oligonucleotide fragments in the mixture away from the originationsite and through a monitoring site remote from the origination site onthe gel. Fluorescent emission from oligonucleotides passing themonitoring point is then detected.

As noted above electrode configurations for applying the potential tothe gel may be of several types. Through these electrodes, a field of upto 350 V/cm, preferably 150-350 V/cm, is applied using a standardelectrophoresis power supply. This high field strength offers theadvantages of rapid migration of the fragments through the gel,resulting in a reduced time to analyze the fragments. The rapidmigration also reduces the impact of diffusion of fragments in the gel,which yields better resolution. Field strengths of this magnitude arenot practically useful in a macrogel, i.e., a gel having a thickness of0.35 mm (350 mm) or greater, because of the enormous current flow andresultant heating that would occur.

FIG. 8 shows one apparatus for use in analyzing oligonucleotidemixtures. In FIG. 8, a light source 41, such as an argon ion laser,shines an excitation beam 40 approximately parallel to the surface ofthe microgel 42. The excitation beam passes through a beam expander 43and an interference filter 44 that removes undesired wavelengths oflight, and then strikes a diffracting mirror 45 selected to reflectlight of the excitation wavelength and transmit light of the wavelengthat which the fluorescent marker emits. The diffracting mirror 45reflects the excitation beam 40 onto the microgel 42. When fluorescentlylabeled oligonucleotides pass through the beam, light is emitted in alldirections, including back towards the diffracting mirror 45. This light46 passes through the diffracting mirror 45, an interference filter 47selected to isolate the emission wavelength from scatter andnon-specific fluorescence, and a lens 48 that focuses the beam 46 on aphotomultiplier tube 49.

FIG. 9 shows an alternative apparatus for analyzing oligonucleotidemixtures in accordance with the invention. As shown, excitation beam 80from light source 81 strikes the surface of the microgel 82 at an anglea, generally from 45 to 65 degrees. Light 86 emitted in the direction ofa photomultiplier tube 89, optionally passing through collection lensesand filters (not shown).

Other apparatus and apparatus configurations may also be used. Forexample, while the apparatus shown in both FIGS. 8 and 9 usephotomultiplier tubes as the detector, other types of detectors such asphotodiodes may also be employed. Photodiodes may be particularly usefulin an apparatus using an array of detectors aligned with the expectedtracks of a plurality of samples on a single gel.

Furthermore, it will be understood by persons skilled in the art thatthe invention is not limited to the use of argon ion lasers as the lightsource. While the argon ion laser is convenient because its emission at488 nm is effective to excite fluorescein, the use of other labels, withdifferent absorption maxima may argue for the use of other type oflasers, or for the use of conventional light sources such as mercury orxenon lamps, combined with appropriate filters.

The microgel holders of the present invention may in principle be usedto form microgels using any type of gel forming material, includingagarose gels. The preferred gel material, however, is a polyacrylamidegel. Polyacrylamide microgels in accordance with the invention, may beformed using either a chemical cross-linking agent catalyst such asTEMED and ammonium persulfate or a photoactivated cross-linking systemsuch as riboflavin and ultraviolet light, or methylene blue and blue(450 nm) light.

Where photopolymerized gels are used in combination with a photocurableadhesive, it may be advantageous to select the gel and adhesive suchthat the polymerization wavelengths for the two systems are different.This flows from the observation that some UV-activated adhesives containmaterials which quench the subsequent curing of UV-cured gels, eitherthrough absorption of the exciting light or by quenching the excitedstates of the gel polymerization initiator. By shifting the gelpolymerization to a different wavelength, this quenching effect isreduced or eliminated.

Prior to the use of a gel for analysis of oligonucleotide fragments, itmay be desirable to pre-run the gel, i.e. to apply a potential gradientwith running buffer, but without the application of a sample. Such apre-run may remove excess reactants from the polymerization reactionwhich could interfere with migration or detection of theoligonucleotides. In addition, since some constituents used in therunning buffer, notably urea, may tend to degrade acrylamide gels, theshelf life of the microgel can be improved by introducing thesecomponents just before the actual running of the analysis.

The use of the invention will now be further illustrated through thefollowing non-limiting examples.

EXAMPLE 1

Gels were prepared using 6% acrylamide (19:1 acrylamide:bis-acrylamide,7M urea, 0.6×TBE) in gel holders formed from 1 mm thick calendaredglass. The bottom plate had a 50 mm gasket surrounding the edge of theplate and forming a central opening 15 mm wide and 75 mm long.Unpolymerized gel was placed in the opening and the top plate placedover it to squash the gel to its desired 50 mm thickness as defined bythe gasket spacer. After polymerization, excess gel was trimmed fromaround the edges of the glass plate.

A test sample was prepared containing a mixture of 0.1 ng/ml of a 30-merhaving the sequence ##STR1## and 0.1 ng/ml of a 31-mer having thesequence ##STR2## These oligonucleotides were synthesized by acommercial supplier of custom oligonucleotides (Oligos Etc.) inaccordance with the inventors' instructions, and were labeled withfluorescein at the 5'-end.

100 nl of the test sample (containing˜100 femtomoles of eacholigonucleotide) was edge loaded onto the top of the gel using acapillary. A field of 100 V/cm was then applied to the gel by laying anelectrode consisting of a 0.25 mm thick strip of indium tin oxide, 35mm×8 mm in size, across each end of the polymerized gel, then connectingthe electrodes to an electrophoresis power supply. An argon ion laserdetection system of the type shown in FIG. 8 was used to detect passageof the fiuorescein-labeled oligonucleotides past a point located 55-65mm from the origin. The signal detected by the photomultiplier tube isshown in FIG. 10. The signal was resolved into two peaks correspondingto the 30-mer (peak A) and the 31-mer (peak B) within a run time of 3minutes.

EXAMPLE 2

A UV activated adhesive matrix was prepared using Minico® M07950-Racrylate adhesive from Emerson & Cuming Inc., Woburn, Mass., mixed with2% by weight Sigma® glass beads (106 micron and finer) filtered toselect beads of a size of 45 to 53 microns. The adhesive matrix wasscreen printed onto the bottom substrate in the pattern shown in FIG. 3.The top substrate was then positioned on top of the bottom substrate asillustrated in FIG. 3. The substrates were then exposed to 20 Watts UVAlight (wavelength 315-385 nm) to initiate curing of the adhesive and tobond the two substrates together.

After the adhesive was cured, the gel holder was placed horizontally ina rectangular filling device of the type described in U.S. patentapplication Ser. No. 08/332,892 and the con-currently filed CIP thereof,which are incorporated herein by reference. Briefly, the microgel holderwas laid horizontally in a quadrilateral filling frame, and placed onthe upper shelf within a filling cabinet. In the cabinet, apolyacrylamide gel forming solution was driven into the gel holder,which was then exposed to ultraviolet light from 20 W UVA-lamps disposedon the interior of the filling cabinet for a period of time sufficientto polymerize the gel.

The gel forming solution used contained 6% acrylamide (19:1bis-acrylamide), 7M urea in 0.6×TBE and 10 ppm riboflavin. Theriboflavin was introduced by adding 10 μl of a 0.4% aqueous riboflavinsolution to 25 ml of acrylamide solution.

EXAMPLE 3

A UV activated adhesive matrix of the type described in Example 2 wasscreen printed onto the bottom substrate in the pattern shown in FIG. 2.One ml of a slurry of 100% ethanol and 1% by weight glass beads (45 to53 microns) was applied to and evenly distri-buted across the bottomsubstrate. After five minutes at room temperature, the top substrate waspositioned over the bottom substrate as illustrated in FIG. 2, and thesubstrates were pressed together. The substrates were then exposed to 20Watts UV lights (315-385 nm) to initiate curing if the adhesive, and tobond the two substrates together to form a gel holder.

Acrylamide solution was injected into the microgel holder andpolymerized as described in Example 2. The force of the injection washedout a portion of the smallest sized glass beads. These beads were easilyremoved from the top of the gel prior to insertion of the LoadingInsert.

EXAMPLE 4

A microgel was prepared as in EXAMPLE 2, using the Insert Panel duringthe polymerization of the microgel. The Insert Panel was carefullyremoved and replaced by the Loading Insert. The microgel holder wasmounted vertically in an electrophoresis apparatus. 3 μl of a preparedsolution containing the products of a completed fluorescein-labeled ddTDNA sequencing reaction of M13 template DNA, with methylene blue andxylenc cyanol as additional visible separation markers, was pipettedinto a loading well.

A TBE solution electrode was formed by gasket sealing the microgelwindow against a TBE reservoir containing a platinum electrode. The"bottom" of the microgel was immersed in a TBE reservoir containing aplatinum electrode. The back of the microgel holder was secured againstan alumina plate by vacuum suction in order to dissipate heat during theelectrophoresis. An electric potential was applied between the twoelectrodes, generating an electric field of 300 V/cm through themicrogel.

Under the clectromotive force, DNA fragments migrated through themicrogel and through the detection zone. An argon ion laser detectionsystem of the type shown in FIG. 8 was used to detect passage of thefluorescein-labeled fragments past a point located 100 mm from theorigin. The signal detected from the ddT reactions by thephotomultiplier tube as a function of time is shown in FIGS. 11A-11F. Asmay be seen in FIGS. 11A-11F. ddT extension products of 1 nucleotidebeyond the primer are observed about 4.9 minutes after the start ofelectrophoresis. Fragments of at least 300 nucleotides may be clearlyresolved within 13.8 minutes from the start electrophoresis.

EXAMPLE 5

A microgel is formed as in Example 2 except that a methylene blueinitiator system is used in place of riboflavin. This initiator systemconsists of 100 μM methylene blue, 1 mM sodium toluene sulfinate and 50μM diphenyliodonium chloride. The gel is polymerized using a source ofblue (450 nm) light such as a quartz-halogen lamp.

EXAMPLE 6

A screen printable low-melting glass composition is prepared bycombining Glass No. 7570 obtained from Ferro Corporation (Cleveland.Ohio) with a binder identified as Product 80 863 and sold by CerdecCorporation (Frankfurt. Germany) in a ration of 10:3 or 1:1, and 1 to10% by weight of solid particles in the form of glass beads prepared asin Example 2 above. Glass No. 7570 has a particles size, D₅₀ of 30.99microns, a thermal expansion coefficient (25°-260° C.) if 82.6×10⁻⁷/°C., a density of 5.36 g/cm³, and a softening point of 449° C. Product80 683 is a water soluble binder used conventionally for application ofglazes to porcelain or ceramics. Other products which might be used inthis capacity include METHOCEL binders available from Dow Chemical.

After mixing the three components of the paste together, the paste isscreen printed onto one glass substrate plate in the desired pattern,for example the patterns shown in FIG. 2 or FIG. 3. The substrate isthen heated to 125° C. for 10 minutes and 320° C. for 10 minutes toprepare the paste for sealing. For sealing, the plate is put in afurnace at 450° C. for 25 minutes to pre-fuse the glass powder. A secondsubstrate plate is then placed on top of the first plate with thepre-fused material sandwiched in between. The whole assembly is thenplaced in a furnace at 470° C. for another 25 minutes, causing finalbinding of the plates together. In order to ensure evenness of the gapbetween the plates, pressure is applied to the outside faces of theplates. This pressure may be generated by clamping, by placing a weightonto the uppermost substrate or using an air driven piston directed fromthe top, downwards against the substrates.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES:2    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:30    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATCGGCTAATCGGCTAATCGGCTAATCGGC30    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:31    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (iii) HYPOTHETICAL: No    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ATCGGCTAATCGGCTAATCGGCTAATCGGCT31    __________________________________________________________________________

What is claimed is:
 1. A method for making an electrophoresis gel,comprising the steps of:(a) applying a plurality of solid particles to asurface of a first substrate; (b) forming a gel holder comprising thefirst substrate, and a second substrate by partially sealing the firstsubstrate to the second substrate such that the plurality of solidparticles is disposed between the first and second substrate, and suchthat a gel compartment having a thickness 250 microns or less is formedbetween the first substrate and the second substrate, said gelcompartment having an opening for the introduction of gel; (c) fillingthe gel compartment with an unpolymerized gel; and (d) polymerizing thegel in the gel compartment, wherein the solid particles have a sizesubstantially equal to the thickness of the gel compartment.
 2. A methodaccording to claim 1, wherein the solid particles are applied to thesubstrate as a mixture with a powdered, low-melting glass furthercomprising the steps ofheating the substrate having the solid particlesand the powdered, low-melting glass thereon to melt the low-meltingglass, and cooling the heated substrate to resolidify the low meltingglass, whereby the solid particles are bonded to the surface of thesubstrate by the resolidified glass.
 3. A method according to claim 1,wherein the first substrate is sealed to the second substrate with anadhesive.
 4. A method according to claim 3, wherein the adhesive isapplied by screen printing.
 5. A method according to claim 3, wherein atleast a portion of the solid particles are dispersed within theadhesive.
 6. A method according to claim 5, wherein the adhesive is anacrylate adhesive.
 7. A method according to claim 1, wherein theseparation between the substrates is from 25 to 100 microns.
 8. A methodaccording to claim 1, wherein the gel is polyacrylamide.
 9. A methodaccording to claim 1, wherein the gel is separated into two or morelanes.
 10. A method according to claim 9, wherein the lanes areseparated by lines of adhesive containing at least a portion of thesolid particles.
 11. A method according to claim 1, wherein at least aportion of the solid particles are disposed on a surface of the topsubstrate or the bottom substrate prior to the formation of the gelcompartment, whereby the plurality of particles are disposed within thegel compartment of the microgel.
 12. A method according to claim 11,wherein the solid particles are disposed on the top substrate or thebottom substrate by application of a slurry of particles in a volatileliquid which is evaporated prior to the formation of the gelcompartment.
 13. A method according to claim 1, wherein the gel isphotopolymerized.
 14. A method according to claim 1, further comprisingthe step of cutting sample introduction and electrode holes in the topsubstrate prior to its assembly into the gel holder.
 15. A methodaccording to claim 14, wherein the sample introduction and electrodeholes are sealed over by a removable adhesive strip prior to the fillingof the gel compartment.
 16. A method according to claim 15, wherein theremovable adhesive strip permits transmission of UV light.
 17. A methodaccording to claim 11, further comprising the step of cutting asubstantially rectangular sample introduction hole into the topsubstrate prior to its assembly into a gel holder, said sampleintroduction hole being formed near a first end top substrate such thatsample loaded through the sample introduction hole will migrate towardsa second, opposite end of the top substrate.
 18. A method according toclaim 17, wherein the sample introduction hole has a long edge closestto the second end of the substrate, and said long edge is beveled suchthat the sample introduction hole has larger dimensions on a firstsurface than on an opposite second surface, and wherein the gel holderis assembled such that the first surface is on the inside of the gelholder.
 19. A method according to claim 18, further comprising the stepof placing an insert into the sample introduction hole prior topolymerization of the gel, wherein the insert is beveled to match thebevel of the sample introduction hole such that a trough is formedbetween the beveled edge of the top substrate and the gel into whichsample may be loaded.
 20. A method for analyzing a mixture of labeledoligonucleotide fragments of different lengths comprising:(a) loadingthe mixture onto an origination site on a microgel, (b) applying apotential to the electrophoresis gel creating an electric field strengthof 100 V/cm or greater, whereby a current flows through theelectrophoresis gel and transports the oligonucleotide fragments in themixture away from the origination site and through a monitoring siteremote from the origination site on the gel; and (c) detectingoligonucleotides passing the monitoring point, wherein the microgelcomprisesa top substrate and a bottom substrate sealed to one another toform a gel compartment having a thickness of from 250 microns or less, aplurality of solid particles having a mean diameter substantially equalto the thickness of the gel compartment, said plurality of solidparticles being disposed within the gel compartment, and anelectrophoresis gel disposed within the gel compartment.
 21. A methodaccording to claim 20, wherein the electrophoresis gel ispolyacrylamide.
 22. A method according to claim 20, wherein at least aportion of the solid particles are dispersed in an adhesive which sealsthe top substrate to the bottom substrate.
 23. A method according toclaim 22, wherein the adhesive is an acrylate adhesive.
 24. A methodaccording to claim 20, wherein at least a portion of the solid particlesare bonded to at least one of the substrates using a low-melting glass.25. A method according to claim 20, wherein the oligonucleotidesfragments are fluorescently-labeled, and are detected as they pass themonitoring point using a fluorescence detector.
 26. A method accordingto claim 25, wherein the fluorescently-labeled oligonucleotides aredetected using an argon ion laser.
 27. A method according to claim 20,wherein an electric field strength of greater than 150 V/cm is created.28. A method according to claim 20, wherein an electric field strengthof from 250 to 400 V/cm is created.
 29. A method according to claim 20,wherein at least a portion of the solid particles are dispersed withinthe gel in the gel compartment.
 30. A loading insert for use in loadinga sample onto an electrophoresis gel, comprising(a) a substantiallyrectangular planar base portion having two long edges and two shortedges and upper and lower faces; (b) a plurality of fingers extendingfrom a first long edge of the base portion in a plane with the upper andlower faces of the base portion, each of said fingers having a width offrom 0.5 to 3 mm; said fingers being evenly spaced at intervals of 2 to7 mm in a region along the first long edge of the base portion startingand ending at a point from 2 to 4 mm from the adjacent short edge of thebase portion; and each of said fingers having a tip portion at thedistal end thereof, said tip portion being cut with a 30°-60° bevelrelative to the plane of the base portion such that the lower surface ofeach finger is longer than the upper surface.
 31. A loading insertaccording to claim 30, wherein the fingers each have a bevel of 45° atthe distal end thereof.
 32. A loading insert according to claim 30,wherein lower surface of the loading insert has a plurality of groovesformed therein, said grooves being parallel to the fingers and extendingacross the entire loading insert.
 33. A filling insert for use infilling an electrophoresis gel holder comprising a substantiallyrectangular planar body member having two long edges and two shortedges, and upper and lower faces, whereina first of said long edges isbeveled at an angle of from 30 to 60 degrees relative to the plane ofthe body member such that the lower surface of the body member is largerthan the upper surface, and a plurality of grooves are formed in the inthe lower surface of the body member, said grooves running parallel tothe short edges and extending from the one long edge to the other.